PROJECT SUMMARY Many solid tumors are unresectable at the time of diagnosis and are associated with very poor survival despite current treatment modalities. Adenoviral vectors are currently the most frequently used vectors for cancer gene therapy. Both a non-replicating p53 expressing adenoviral vector and a replication selective adenovirus (H101) have received regulatory approval in China. The use of replicating adenoviruses is a logical development in that by repeated rounds of infection, release and re-infection of adjacent tumor cells, the virus might be anticipated to spread from cell-to-cell through the tumor mass. The success of recombinant adenoviruses as a cancer therapy is, however, limited by sub-optimal oncolytic activity and inefficient distribution of the virus within the tumor mass. The objective of this proposal is to introduce and evaluate several modifications, that we have previously constructed and validated individually, into the same viral backbone. These modifications are designed to be complementary and have the potential to dramatically increase the oncolytic activity of the virus to a level that far exceeds the activity of viruses currently in clinical trials. In the previous funding period it was shown that MMP-8 gene expression from a non-replicating adenovirus breaks down tumor collagen stroma and markedly increases tumor responses induced by the wild type adenovirus. In the first aim MMP-8 will be introduced into a replicating virus and it will be determined if a replicating adenovirus that expresses MMP-8 is highly effective at tumor cell killing. It will also be determined if transcriptional control of a modified E1a gene will target replication of an MMP-8 expressing adenovirus to tumor cells. In aim 2 it will be established if MMP-8 expression has adverse effects that could be mitigated by tumor specific targeting. In aim 3, it will be established if viral mediated p53 gene expression can improve the oncolytic activity of a targeted MMP-8 expressing replicating adenovirus, and if this can be further improved by chemotherapy. To ensure optimal activity p53 will be expressed late in the viral life cycle and E1a will be modified to prevent binding to p300 that may impair p53-mediated transactivation. The E1b19kD gene will be deleted to improve apoptosis induction, and a p53 modification will be introduced to block viral E1b-55kD and/or cellular mdm2 mediated p53 degradation. In aim 4 tumor responses achieved by a replicating adenovirus with modified E1a gene and E1b19kD gene deletion that expresses p53 and MMP-8 transgenes will be evaluated in combination with current therapies. As a model system for this aim we have chosen lung cancer, and will evaluate the combination of virotherapy with standard chemotherapeutic and angiogenesis-modifying regimens. Efficacy and safety studies will also be performed in a syngeneic hamster model. Recently, it has been reported that the human adenovirus will replicate in the Syrian Hamster making this an ideal model for safety and efficacy evaluation.